Elsevier

Clinical Immunology

Volume 140, Issue 1, July 2011, Pages 102-116
Clinical Immunology

Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2nullIl2rγnull mouse model: A multivariable optimization approach

https://doi.org/10.1016/j.clim.2011.04.002Get rights and content

Abstract

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2nullIl2rγnull strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34 cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2nullIl2rγnull humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.

Graphical abstract

Research Highlights

► Improving human hematopoietic chimerism in a BALB/c humanized mouse model. ► Test culture conditions of CD34+ and co-injection of CD34− cells. ► Test route of injection, and CD34+ cells from PBL. ► Found higher chimerism culturing CD34+ cells 1 to 8 days, co-injecting CD34− cells. ► Negative effects from culture with IL-3 and with CD34+ cell from PBL.

Introduction

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice, are becoming an important tool for studying the development and function of the human immune system (HIS). Major advances in the engraftment of a HIS in mice have been recently achieved through the use of novel recipient mice that bear mutations in the IL-2 receptor common gamma chain gene (Il2rγ) in combination with either the deletion of one of the recombination activating genes (Rag1 or Rag2) or the naturally occurring Prkdc-scid mutation [1]. Together these mutations lead to the development of mice with a largely dysfunctional immune system lacking B, T and NK cells, and allowing better xeno-engraftment [1], [2], [3]. The genetic background of the immunodeficient mouse strains is also known to contribute to HIS engraftment. The BALB/c [4], [5], [6] and the NOD [2], [3], [7], [8] represent successful recipients for HIS, while the C57BL/6 genetic background retains immunorejection capabilities [1]. Recent studies, however, have indicated that the NOD immunodeficient strain may be a better recipient for the development of a HIS than the BALB/c strain [9].

Although the increased human hematopoietic engraftment achieved with these novel immunodeficient recipient strains is currently a major advance, the model is still not optimized for many applications. A major obstacle is the large variability of human engraftment among recipients generated with HSCs isolated from different or even the same donors [4], [5], [7], [8]. This variability also extends to the type of human hematopoietic cells and their products. Furthermore, the stability of the human grafts is also variable, with many recipients losing chimerism over time while others maintain long-lived grafts that can be transferred to secondary recipients [7], [10]. This variability likely reflects differences in the potency and homing abilities of HSCs, as well as in their longevity. Understanding of human HSC biology has improved through research aimed at increasing the efficiency of bone marrow, umbilical cord blood and mobilized peripheral blood transplantation in the clinic. For instance, although the CD34 antigen is commonly accepted as a primitive hematopoietic stem cell marker, subsets within the CD34+ cells exist and display varying degrees of engraftment potential [11], [12], [13], [14], [15], [16]. In addition, effort has focused on expanding HSCs in culture, testing the effect of cytokines and media in this context [17], [18]. Thus, a more refined definition of hematopoietic stem cell phenotype and better conditions for HSC manipulations may improve experimental consistency in the generation of humanized mice.

The method to generate humanized mice varies greatly between investigators. Human HSCs are injected into either adult or newborn recipients, with the idea that young animals have a better tolerance to xenografts and/or more receptive tissue [9], [19]. Additionally, HSCs are transplanted intra-venously (i.v.), intra-hepatically (i.h.), intra-peritoneally, intra-splenically, intra-cardiacally (i.c.), or directly into the bone marrow [4], [7], [9], [10], [20], [21], [22], with the notion that certain tissue microenvironments may increase the engraftment of these cells. Moreover, human HSCs are most commonly isolated from umbilical cord blood, but can also be obtained from fetal liver, adult bone marrow or G-CSF-mobilized peripheral blood [3], [6], [23], [24]. Finally, donor cells are injected either fresh or after freezing, and are often expanded in vitro for days or even weeks with different cytokine cocktails [11], [25]. It is often unclear how the methods for the generation of these mice affect their ultimate phenotype. Here, we performed a study aimed at optimizing a protocol for the generation of hematopoietic humanized mice displaying increased development and consistency of human hematopoietic chimerism and of human lymphocytes. Our study is based on the use of BALB/c-Rag2nullIl2rγnull (hereafter referred to as BALB/c-DKO) neonate mice as recipients of umbilical cord blood-derived HSCs [4], [5]. Parameters analyzed in this study were the injection route and culture conditions of CD34+ HSCs, and whether these cells could be frozen for future use. In addition, we tested whether co-injection of CD34 human cells supported the engraftment and differentiation of CD34+ HSCs by potentially providing factors that are important for the development and survival of human hematopoietic cells.

In accordance with previous reports, our data show the existence of a large variability in the rate and degree of human hematopoietic engraftment in humanized mice. However, we show that certain conditions significantly increase the numbers of engrafted mice as well as the degree of human leukocyte engraftment and the generation of human B and T cells, indicating that humanized BALB/c-DKO mice represent a useful animal model for the study of a human immune system.

Section snippets

CD34+ and CD34 cell preparation from human umbilical cord blood

Umbilical cord blood units were obtained from the University of Colorado Cord Blood Bank at ClinImmune Labs (Aurora, CO) as samples that were rejected due to low volume. Investigators in this study were blinded from donor identities, and the studies were performed in compliance with the National Jewish Health (NJH) Institutional Review Board. Blood mononuclear cells were isolated over Ficoll-density gradients. CD34+ cells were enriched using human CD34-specific magnetic beads and an AutoMACS

Experimental design

In this study we used the BALB/c-DKO newborn model as a recipient of hHSCs [4]. Human HSCs were isolated from umbilical cord blood, although some experiments using HSCs recovered from untreated adult peripheral blood were also performed (see supplemental data). The number of HSCs injected ranged from 5 × 104 to 2.1 × 106 cells/mouse, and varied based on number of pups available for injection at any given time and whether the cells were cultured. The lower range is consistent with the literature

Discussion

Our study was performed to test parameters that potentially affected the survival, engraftment and/or differentiation capacity of umbilical cord blood-derived hHSCs in immunodeficient mice, and to generate a protocol optimized for the establishment of humanized mice in the BALB/c-DKO newborn model. We show that culturing hHSCs for 1 to 8 days in the presence of IL-6, SCF, and FL, and co-injecting these cells with T cell-depleted CD34 cells into sublethally irradiated newborn BALB/c-DKO mice,

Conclusions

Our study used a multi-parameter approach to setup an optimized protocol for the generation of humanized mice in the BALB/c-Rag2nullIL2rγnull strain. This protocol results in higher levels and lower variation of human hematopoietic engraftment in mice, allowing easier comparison of human immune systems between mice generated with different hHSC samples.

The following are the supplementary materials related to this article

. Freezing HSCs does not compromise their engraftment and differentiation in

Acknowledgments

This study was supported by the National Institute of Health with the R21-AI073629 grant, and by Arthritis Foundation with an Innovative Research Grant. The BALB/c-Rag2nullIl2rγnull (BALB/c-DKO) mice were a generous gift from Dr. Irving Weissman (Stanford University, CA). We are deeply thankful to the ClinImmune Labs for providing us the umbilical cord blood samples for our studies. We thank Drs. James Murphy and Doug Everett (NJH) for assisting with statistical analyses, and Dr. Sarah Rowland

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