Busulfan som en myelosuppressiv Agent for generering Stabilt på høyt nivå Bone Marrow kimerisme i Mus

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. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the β-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays.

For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells^2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called ‘lethal irradiation’). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections³. As such, reduced intensity conditioning (so called ‘RIC’ protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly⁴.

Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation^5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab⁷, as well as in several other recent publications^8–11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM⁷. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation⁷.

We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)⁷ and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS^7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders^7–11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning⁷, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.

While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

Protocol

MERK: Etikk Uttalelse: Denne protokollen er gjennomgått og godkjent av Animal Care komité Simon Fraser University (UACC; tillate tall 1037K-12 og 1060K-03) og er i samsvar med den kanadiske Council on Animal Care, NIH guide for Stell og bruk av forsøksdyr, og EEC direktiv. 1. Spesielle hensyn MERK: Busulfan er cytotoksiske. Håndter og kast i henhold til sikkerhetsdatablad og institusjonelle retningslinjer. Utføre alle teknikker aseptisk i en lamin…

Representative Results

Administrasjon av 60-100 mg / kg av busulfan er generelt godt tolerert av mus. Men dyrene vanligvis gå ned i vekt i løpet av kondisjoneringsfasen (~ 5-10%) og dermed dietten kan trenge å bli supplert med godbiter som bestråles solsikkefrø for å oppmuntre spise. Nivåer av kimerisme på> 80% GFP + celler i perifert blod og BM bør være gjennomgående oppnåelig ved bruk av 60 til 100 mg / kg busulfan 7. Vårt laboratorium har med hell brukt denne protokollen for å generere over 100 kimære mus med&g…

Discussion

Busulfan kondisjone muliggjør generering av høyt nivå BM kimerisme i mus med hematopoetiske celler som er lett detekterbart. Denne kondisjonering kan utføres uten behov for bestrålingsanlegg. Videre er myelosuppresjon med de doser av busulfan skissert i denne protokollen godt tolerert, minimaliserer toksiske bivirkninger forårsaket av myeloablative doser av bestråling, og kan således være en mer egnet teknikk for å generere BM kimerisme i unge, gamle eller syke mus som er mer utsatt for myeloablativ bestrålin…

Materials

Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
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