Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Summary

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Abstract

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism >80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

Introduction

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis¹. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals¹, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. T....

Protocol

NOTE: Ethics Statement: This protocol has been reviewed and approved by the Animal Care Committee of Simon Fraser University (UACC; permit numbers 1037K-12 and 1060K-03) and is in compliance with the Canadian Council on Animal Care, the NIH Guide for the Care and Use of Laboratory Animals, and the EEC Council Directive.

1. Special Considerations

NOTE: Busulfan is cytotoxic. Handle and dispose according to the material safety data sheet and institutional guidelines.

1. Perform all techniques aseptically in a laminar flow hood.
2. Sterilize instruments prior to use by wrapping in an appropriate pack....

Results

The administration of 60-100 mg/kg of busulfan is generally well tolerated by mice. However, animals typically lose weight during the conditioning phase (~5-10%) and thus the diet may need to be supplemented with treats such as irradiated sunflower seeds to encourage eating. Levels of chimerism of >80% GFP+ cells in the peripheral blood and BM should be consistently achievable using 60-100 mg/kg busulfan⁷. Our laboratory has successfully used this protocol to generate over 100 chimeric mice with >80% GFP.......

Discussion

Busulfan conditioning allows for the generation of high-level BM chimerism in mice with hematopoietic cells that are readily detectable. This conditioning can be performed without the need for irradiation facilities. Moreover, myelosuppression with the doses of busulfan outlined in this protocol is well tolerated, minimizing toxic side effects caused by myeloablative doses of irradiation, and thus may be a more suitable technique to generate BM chimerism in young, old, or diseased mice that are more susceptible to myeloa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Busulfex	Otsuka Canada Pharmaceutical Inc.	DIN 02240602	Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.
ACK Lysing Buffer	Life Technologies	A10492-01	Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly
Fetal Bovine Serum	Life Technologies	12483-020