Center for Regenerative Medicine, MGH - Massachusetts General Hospital
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Yusuf, R. Z., Scadden, D. T. Homing of Hematopoietic Cells to the Bone Marrow. J. Vis. Exp. (25), e1104, doi:10.3791/1104 (2009).
Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.
Homing is the process whereby bone marrow cells including stem cells, progenitor cells and differentiated cells, their way into the bone marrow after being injected into the blood stream or bone marrow cavity of mice. As is obvious from the definition, a scientist may chose to study the homing of hematopoietic stem cells, progenitor cells or differentiated cells identified by immunophenotyping and isolated by cell sorting. Typically scientists inject bone marrow cells depleted for lineage markers and hence enriched for progenitor and stem cells.
The dose of the cells of interest injected is a variable in this experiment. The number of cells can vary from 500 000 per recipient mouse to 20 million per recipient mouse. The more cells one can inject the easier the detection in the bone marrow. Cell number availability may limit the number injected. Sorting 20 million LKS SLAM cells is extremely difficult perhaps even impractical with the current technologies in most laboratory settings, in which case one might decide to use a number of cells towards the lower end of the range given above.
The hose animal in which homing is being studied can also be varied. This recipient animal may be a WT C57BL/6J mouse which has been lethally irradiated with 9 Grays of radiation as we did in this experiment, a WT C57BL/6J mouse or a W/Wv mouse which has not been irradiated. Whether the mouse is irradiated or not depends on the question the experimenter is trying to answer. If one is interested in studying homing in a setting where a large amount of cytokines is being released and vascular leak is being induced, an irradiated WT mouse is an appropriate choice. A smaller number of cells (500 000) may be injected into a WT mouse which has not been irradiated so that the stem cells can home to a small number of unoccupied niches without the above confounders.
W/Wv mice are deficient in c-KIT receptor function and can engraft stem cells without any preconditioning. This allows us to study the homing of stem cells in the absence of vascular injury or cytokine release caused by radiation. The logistic problem with the use of these mice is that it is difficult to obtain adequate numbers of WWv mice to serve as recipients due to breeding colony size concerns.
In brief, although we describe a standard homing protocol, the variations used depend upon the question to be answered and must be determined during the design of the experiment.
This protocol was tested and optimized in the laboratory of Dr. David Scadden. Funding is provided by the Dana Farber Cancer Institute/Massachusetts General Hospital Clinical Hematology and Oncology Fellowship Program.
|DPBS (Dulbecco’s Phosphate Buffered Saline||Mediatech, Inc.||21-031-CV|
|Sterile Fetal Bovine Serum||Valley Biomedical, Inc.||BS3033|
|0.5M EDTA (pH 8.0)||Boston BioProducts||BM-150|
|Tryphan Blue solution||Mediatech, Inc.||25-900-CI|
|ACK Lysing Buffer||Lonza Inc.||10-548E|
|Isopropranolol U.S.P.||Denison Pharmaceuticals, Inc|
|Vybrant DiD cell-labelling solution||Invitrogen||V22887|
|Vybrant DiI cell-labelling solution||Invitrogen||V22885|