Immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development

Abstract

Immunodeficient mice engrafted with human immune systems provide an exciting model to study human immunobiology in an in vivo setting without placing patients at risk. The essential parameter for creation of these "humanized models" is engraftment of human hematopoietic stem cells (HSC) that will allow for optimal development of human immune systems. However, there are a number of strategies to generate humanized mice and specific protocols can vary significantly among different laboratories. Here we describe a protocol for the co-implantation of human HSC with autologous fetal liver and thymic tissues into immunodeficient mice to create a humanized model with optimal human T cell development. This model, often referred to as the Thy/Liv or BLT (bone marrow, liver, thymus) mouse, develops a functional human immune system, including HLA-restricted human T cells, B cells, and innate immune cells.

Figure 1. Surgical procedure for implantation of human fetal thymus and liver

(A) Fur is removed from anesthetized NSG mice and (B) a 0.5-cm incision in a longitudinal direction in the abdominal wall (through muscle wall). (C) Using a pair of straight micro-dissecting forceps, expose kidney through the incision. (D) Secure the kidney by grasping the renal vessels and ureter just below kidney between the flaps of skin with a pair second pair of straight micro dissecting forceps. Make a small (1 to 2 mm) incision in kidney capsule at the posterior lateral side with a scalpel. Insert the trocar loaded with human tissue as far as possible through the incision in the kidney capsule and staying as superficial as possible. Push the plunger piece of the trocar to expel the loaded tissue. (E) Remove trocar from kidney. Gently pull apart the two cut edges of the muscular wall using two pairs of micro dissecting forceps. The kidney will retract into the peritoneal cavity. (E) Close the abdominal wall with two separate sutures, using the Olsen-Hegar needle holder and surgical suture. Cut the ends of the suture and close the knots. Close the skin incision with three autoclips, using an autoclip wound-clip applicator. (F) Mice were then placed on a warming tray and observed until they awoke from the anesthesia.

Figure 2. Evaluation of human cell chimerism

At 12 weeks post-implant human immune cell chimerism levels can be evaluated in the peripheral blood. Our standard panel includes antibodies specific for mouse CD45, human CD45, human CD3, human CD20, human CD4 and human CD8. (A) The gating strategy for this panel includes first evaluating percentages of human CD45+ and mouse CD45+ cells. (B) Human CD45+ cells are evaluated for expression of human CD3 to enumerate T cells and human CD20 to enumerate B cells and (C) then the T cells populations are further defined by CD4 and CD8 expression. (D) In addition the human thymocytes recovered from the thymic organoid can be evaluated by expression of human CD4 and CD8.