Parameters for establishing humanized mouse models to study human immunity: Analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγ^null mutation

Abstract

“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγ^null mutation; NOD-scid IL2rγ^null, NOD-Rag1^null IL2rγ^null, and BALB/c-Rag1^null IL2rγ^null mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.

Introduction

The discovery of the severe combined immunodeficiency mutation (Prkdc^scid, abbreviated as “scid”) in a stock of C.B-17 mice in 1983 [1] led to the assessment of their ability to support engraftment with human peripheral blood mononuclear cells (PBMC, termed Hu-PBL-SCID) [2] or fetal (termed SCID-Hu) [3] or adult (termed Hu-SRC-SCID) [4] hematopoietic stem cells (HSC). These human hematolymphoid engrafted mouse models have been used in multiple biomedical disciplines for the study of human immunobiology and hematopoiesis (reviewed in refs. [5–8]). However, the utility of the early strains of immunodeficient mice transplanted with human hematolymphoid cells was hindered by the inability to achieve multilineage hematopoietic engraftment leading to the generation of a functional human immune system [5], [6]. In the Hu-PBL-SCID model, although all lineages of peripheral blood mononuclear cells (PBMC) are injected, the majority of human cells engrafted are T cells, with few B cells, myeloid cells or natural killer (NK) cells surviving. In the Hu-SRC-SCID model, the major obstacle was the inability of HSC-engrafted mice to develop all components of a functional human immune system, particularly T cells [5].

A major technological breakthrough occurred when investigators created genetic stocks of scid, Rag1^null or Rag2^null mice that also harbor mutations in the IL2 receptor common gamma chain (IL2rγ) gene [9], [10], [11], [12]. IL2rγ is required for high-affinity signaling through multiple cytokine receptors, including IL2, IL4, IL7, IL9, IL15 and IL21 [13]. Immunodeficient mice bearing a mutated IL2rγ gene support much higher levels of human hematolymphoid engraftment than all previous immunodeficient stocks. Multiple immune lineages develop following engraftment of human HSC that can lead to the generation of a functional human immune system [5], [6], [7], [8].

Confounding widespread establishment of humanized mouse models in multiple laboratories is the diversity of approaches that have been reported for engrafting human HSC. For example, IL2rγ^null mice are available on a number of strain backgrounds. The ability of immunodeficient mice to support engraftment of human hematolymphoid cells has been shown to be strongly affected by the genetic background of the host [5], [14], [15], [16], [17]. In addition to strain background, reports differ significantly with regard to engraftment methodologies, which include intravenous (IV) engraftment into adult mice [11], or intrahepatic (IH) [10], intraperitoneal (IP) [12], and IV [9], [18] injection into newborn mice.

In the present study, we compared a number of variables of human HSC engraftment, including strain background, age of recipient, and engraftment route. The three strains of immunodeficient mice tested were NOD-scid IL2rγ^null, NOD-Rag1^null IL2rγ^null, and BALB/c-Rag1^null IL2rγ^null. To provide a consistent comparison, we used human HSC derived from umbilical cord blood (UCB) in all of our experiments. We matched UCB donors within an individual experiment, allowing direct comparison of the engraftment characteristics in each strain, independent of donor-to-donor variability. We observed that in the variables tested, newborn immunodeficient NOD strain mice provided the optimal hosts with respect to their ability to support human hematopoietic cell engraftment.