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New Blood

Researchers are now discovering unexpected activities of blood stem cells by stu

Researchers are now discovering unexpected activities of blood stem cells by studying them during infection.

By Christopher Hall & Philip Crosier

Chemotherapy takes a huge toll on the immune system, but new research into blood stem cell proliferation could improve the recovery of patients.

Blood stem cells are rare cells that maintain appropriate numbers of mature blood cell types throughout life. They are important because they can replace the entire blood system when transplanted into patients with blood disorders. Identifying new ways to increase the number of these powerfully regenerative stem cells is an area of intense research.

Recent studies have revealed that the numbers of these cells increases during infection. This has led researchers to try and understand how the body instructs its pool of blood stem cells to expand in response to infection.

By observing blood stem cells within live infected zebrafish larvae, we have identified a new genetic pathway that drives the expansion of these valuable cells. Identifying the genetic pathways controlling this response may enable researchers to increase the numbers of stem cells in the laboratory, thus increasing their regenerative potential when administered to patients.

Stem Cell Regeneration after Leukaemia Therapy

Stem cells have the remarkable ability to develop into different specialised cell types. In many tissues, limited numbers of stem cells provide a type of internal repair system, replacing cells lost as a result of normal wear and tear, injury or disease.

Mammalian blood stem cells, whose numbers are established during early development, reside in the bone marrow and are responsible for establishing and maintaining the entire blood system throughout life. Blood stem cells, like other adult stem cells, remain in a largely inactive state and rarely divide. When they do divide, each daughter cell has the potential to remain a stem cell or eventually become a specialised blood cell type. These regenerative qualities have clinical applications, particularly involving disorders of the blood system.

One such example is in helping the blood system recover following chemotherapy treatment of leukaemia. Many chemotherapeutic drugs act by killing rapidly dividing cells. But while this eliminates cancer cells, it also causes extensive collateral damage to the entire blood system, with large numbers of dividing blood cells also killed. Blood stem cell transplantation is sometimes used to help the blood system regenerate following chemotherapy.

There is much interest in understanding what controls blood stem cell proliferation, as manipulating these processes in the laboratory holds considerable promise for enhancing their use in transplantation.

The Lens of Infection

Much of our understanding about blood stem cells has come from examining their activity during normal physiological conditions. Researchers are now discovering unexpected activities of blood stem cells by studying them during infection.

Neutrophils are a specialised type of immune cell that primarily destroys pathogenic bacteria. However, large numbers of neutrophils perish as a result of their normal response to infection. This necessitates a replenishment strategy for the host to maintain adequate neutrophil numbers and immune protection.

Investigating the signalling pathways controlling immune cell replenishment during infection has led to the unexpected observation that blood stem cells are directly responsive to infection. It is now recognised that blood stem cells proliferate as part of the normal response to infection, thus replenishing immune cells such as neutrophils.

Much remains unknown about the molecular mechanisms controlling these processes. The prospect of manipulating newly identified signalling pathways to activate blood stem cells is an area of intense research interest.

What We Found

Having research interests in how neutrophils function during infection and how blood stem cells develop, we sought to generate a transgenic zebrafish that would enable us to image these cells within live zebrafish. To achieve this we created transgenic zebrafish that possess fluorescently marked neutrophils and blood stem cells. By simply looking under a special fluorescent microscope we could then directly observe these cells and their behaviour within transparent embryos and larvae.

Using transgenic zebrafish embryos with marked neutrophils, we were among the first groups to capture how neutrophils behave in response to bacterial infection and during the wound-healing process within an intact whole-animal system. And by employing transgenic embryos with marked blood stem cells, we were able to directly image the embryonic origins of blood stem cells. In doing so we helped resolve a long-standing controversy regarding the origin of the blood stem cell.

When live-imaging the immune response of fluorescent neutrophils to bacteria we noticed that many infected zebrafish larvae, upon resolving the infection, became almost completely depleted of neutrophils. Immediately following this we could see larval blood-forming domains become progressively populated with neutrophils in much the same way as occurs in mammals.

Notably, these newly emerging neutrophils populated the site where we had previously observed the first blood stem cells to emerge. Our previous studies of blood stem cell emergence within zebrafish embryos had shown that blood stem cells “budded off” from specialised cells located in the wall of the embryonic trunk aorta. They then populated a region of the embryonic trunk between this arterial vessel and an underlying vein.

The timing of neutrophil replacement after infection coincided with the period when the region between the major trunk vessels contains blood stem cells. This suggested to us that the newly emerging neutrophils were developing from these blood stem cells, and suggested that blood stem cells were somehow sensing the infection and altering their activity to produce more neutrophils.

We next wanted to examine how the infection event (in the head of the developing zebrafish) was signalling to this anatomically separate blood stem cell region to produce more neutrophils. To our surprise we observed that there was an increase in the number of blood stem cells compared with non-infected control larvae.

The free radical nitric oxide (NO) is an important cellular signalling molecule that plays many different roles during both physiological and pathological processes. The production of NO is controlled by a family of enzymes called nitric oxide synthases (NOS) that catalyse a reaction generating NO from the amino acid L-arginine.

Coincident with our discovery that the zebrafish blood stem cell compartment expanded in response to infection, an elegant study reported a role for one of the NOS enzymes as a positive regulator of zebrafish blood stem cell development. Importantly, this role was later confirmed within mice, thus demonstrating that its function was conserved within mammals.

Of interest is that one of the NOS genes, inducible NOS (iNOS), is expressed following infection. This prompted us to evaluate a role for iNOS during blood stem cell expansion following infection.

Using the zebrafish model we discovered that iNOS was required for the infection-responsive increase in blood stem cell numbers. Further studies revealed that this iNOS activity depended on another infection-responsive gene called C/EBP-beta that controlled the expression of iNOS within blood stem cells.

What Next?

The litmus test regarding whether this discovery has therapeutic potential is to assess if a similar infection-responsive mechanism operates for human blood stem cells and whether it can be manipulated for therapeutic benefit. Further studies will also be necessary to understand precisely how C/EBP-beta-driven iNOS instructs dormant blood stem cells to proliferate.

Ultimately, identifying drugs that can activate this infection-responsive capacity of blood stem cells to proliferate could prove therapeutically useful for blood cell expansion and transplantation applications, such as following chemotherapy treatment of leukaemia.

Christopher Hall is a Senior Research Fellow and Philip Crosier is a Professor of Molecular Medicine at The University of Auckland’s Department of Molecular Medicine and Pathology.