Troubleshooting: Quantification of mobilization of progenitor cell subsets from bone marrow in vivo.
BACKGROUND
The molecular mechanisms that control the mobilization of specific stem cell subsets from the bone marrow are currently being intensely investigated. It is anticipated that boosting the mobilization of these stem cells via pharmacological intervention will not only produce more effective strategies for bone marrow transplant patients, but also provide novel therapeutic approaches for tissue regeneration.


METHODS
Measurement of stem cell mobilization by sampling peripheral blood is problematic because it is technically difficult to accurately determine absolute numbers of rare progenitor cells by blood sampling. Furthermore a rise in progenitors may be caused by release of stem cells from tissues other than the bone marrow (e.g. spleen and adipose), or indeed an inhibition of stem cell homing back to the bone marrow or other tissues. Finally it is not possible to distinguish whether the pharmacological agent is acting directly at the level of the bone marrow or mobilizing progenitors by a distinct indirect mechanism. To resolve these problems, we have developed a technique that allows perfusion of the vasculature of the hind limb bone marrow in situ in mice. In this system, the femoral artery and vein are cannulated in situ such that the femur and tibia bone marrow are perfused in isolation under anaesthesia. As such, pharmacological agents can be administered directly into the bone marrow vasculature. Mobilized cells are then collected via the femoral vein and colony assays performed in defined growth media to allow identification of haematopoietic, endothelial, and mesenchymal progenitor cells. We have used this system to determine the ability of a CXCR4 antagonist to mobilize these distinct types of progenitor cells from the bone marrow of mice pre-conditioned with either G-CSF or VEGF.


CONCLUSIONS
This isolated hind limb perfusion system has allowed comparisons to be made between cytokines (G-CSF and VEGF) that act chronically, either alone or in combination with agents that act acutely on the bone marrow (CXCR4 antagonist) on their ability to directly mobilize specific populations of stem cells. Data obtained therefore gives a more accurate understanding of the efficacy of different mobilizing strategies compared to peripheral blood analysis.
Sackler Institute of Pulmonary Pharmacology, Division of Pharmaceutical Sciences, King's College London, SE1 9NH, UK. simon.pitchford@kcl.ac.uk
Simon C
SC
Pitchford
Mark J
MJ
Hahnel
Carla P
CP
Jones
Sara M
SM
Rankin
D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000502:physiology*; D002452:Cell Count; D003584:Cytological Techniques / Q000592:standards; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods* / Q000592:standards; D006614:Hindlimb / Q000098:blood supply / Q000502:physiology; D006801:Humans; D051379:Mice; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors; D012039:Regional Blood Flow / Q000502:physiology; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D019718:Receptors, CXCR4; D042461:Vascular Endothelial Growth Factor A; D016179:Granulocyte Colony-Stimulating Factor