Fetal liver CD34⁺ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice

Background: Transplantation of CD34⁺ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34⁺ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34⁺ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34⁺ isolated from cord blood (CB-CD34⁺) or fetal liver (FL-CD34⁺ and FL-CD34⁺CD14^-) and their engraftment into immunocompromised NOD/Shi-scid Il2rg^null (NOG) mice.

Methods: NOG mice were transplanted with either CB-CD34⁺, FL-CD34⁺ or FL-CD34⁺CD14^- to generate CB-NOG, FL-NOG and FL-CD14^--NOG, respectively. After 15-20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles.

Results: We show that CB-CD34⁺ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34⁺ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34⁺ isolates contain non-hematopoietic CD14⁺ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14⁺CD34⁺ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14⁺CD34⁺ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection.

Conclusion: Our results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.