Generation of Bone Marrow Chimeras

Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Abstract

Bone marrow chimeras are widely used in immunological studies, to dissect the contributions of hematopoietic and non-hematopoietic cells in immune cell development or functions, to quantify the impact of a given mutation, or in preclinical studies for hematopoietic stem cell transplantation. Here we describe a set of procedures for the generation of bone marrow chimeras.

1. Introduction

Immune and blood cells develop from hematopoietic stem cells (HSC) located in the bone marrow or fetal liver, in adult and fetal mice, respectively. Because HSC, the immature progenitors, and most mature cells derived thereof are more radio-sensitive than most other tissues, including non-hematopoietic thymic stromal components, it is possible to use irradiation doses consistent with animal survival that ablate the immune and hematopoietic system. Developmental “niches” emptied by radiation-induced myeloablation can be filled by HSC transplanted by intravenous injection, generating chimeric animals in which most or all hematopoietic and immune cells are derived from donor-derived HSC, whereas non-hematopoietic cells remain of host origin. The generation of bone marrow chimeras, on which this chapter focuses, has been a powerful tool to study T cell development, and continues to be invaluable to study a variety of immune processes and immune cell functions [1]. In addition to its use in biomedical research, HSC transplantation is used routinely in the clinic, with indications spanning from treatment of genetic blood disorders to cancer therapy; experimental bone marrow chimeras are important preclinical tools for such studies, although this aspect will not be addressed further here.

Despite its broad applicability, bone marrow chimera generation is inefficient for studies of specific cell types that develop exclusively or principally from fetal HSC or to study the development of cells from mutant mice that carry mutations incompatible with postnatal development or survival. Such situations can benefit from the generation of fetal liver chimera that use a similar procedure to transplant fetal HSC [2] and is not described here.

In the specific field of T cell development studies, HSC transplantation is mostly used in two distinct circumstances. First, it can distinguish, among the phenotypic consequences of a given mutation, those that are intrinsic to the hematopoietic component of the thymus (generally the developing thymocytes) and those that primarily affect the host thymic environment, especially the thymic epithelium. Specific applications of this approach study the impact of various signaling components of the thymic epithelium, including MHC haplotypes, antigenic peptides, and stromal cytokines, on thymocyte development. Second, “mixed” bone marrow chimera, in which irradiated hosts are reconstituted with HSC of two distinct genotypes (e.g., mutant and wild-type) are highly sensitive tools to identify and quantify incomplete developmental defects. Varying the respective proportions of each component is useful to identify indirect effects [3].

Although the generation of bone marrow chimeric mice does not involve any particularly challenging step, it requires careful logistics for animal procedures and a specialized infrastructure, especially for irradiation. After transplantation, myeloid and B cell reconstitution precedes that of the T cell compartment, which is not complete before 8 weeks, even though donor-derived thymocytes can generally be detected 3–4 weeks after transplantation [1].

Because usual myeloablative irradiation doses do not completely ablate the host immune system, allelic markers are used to distinguish donor from recipient cells at time of analysis. Commonly used surface molecules for this purpose are CD45 or CD90 (Thy-1). Congenic mice carrying variants of these molecules recognized by allele-specific antibodies (e.g., CD45.1 and CD45.2 or CD90.1 and CD90.2) are available from commercial providers. Despite their names, genetic differences between such “congenic” lines and the reference strain (e.g., C57BL/6) are extensive and often include genes not genetically linked to the locus encoding the allelic markers. Recent studies have shown the potential consequences of such extensive variations [4], and new mouse strains are being developed to address these issues. Transgenic expression of fluorescent protein, e.g., green fluorescent protein (GFP), can be used as an alternative to track donor-derived versus host cells.

2. Materials

2.1. Preparation of Recipient Mice (See Note 1)

8–12-week-old recipient mice (e.g., C57BL/6 congenic CD45.1), preferentially gender matched to donor mice (see Notes 2 and 3).