Reconstruction of Megaloblastic Anemia in a Novel Erythroblastic Island Humanized Mouse Model with Mature Circulating Human Red Blood Cells

Human immune system (HIS) mice are powerful tools for human hematopoietic system research but lack efficient reconstitution of human red blood cells (hRBCs) as a result of peripheral clearance by murine macrophages. Administration of clodronate liposome (Clod) abrogates murine macrophages and rescues circulating peripheral blood (PB) hRBCs in HIS mice, but only transiently and with significant toxicity. In 2021, liver and cytokine double humanized huHepMISTRG-Fah model destructs murine liver macrophages and rescues circulating hRBCs. However, the maturation status of hRBCs in PB of this model is not comparable with that of human counterparts, as indicated by the abnormal expression of human CD71. Consequently, the development and evaluation of therapeutics for maturation-associated erythropoietic diseases in HIS mice, such as hereditary megaloblastic anemia (MgA), is severely hindered by the maturation abnormality of hRBCs.

Previous studies focused on the erythro-phagocytotic function of murine macrophages, thereby overlooking their supporting role in the erythropoiesis. The differentiation of erythroblasts is accompanied by the formation of erythroblastic islands (EBIs), which are described as a macrophage surrounded by erythroblasts of different maturation stages. The macrophage within the EBI is involved in nucleus digestion, iron transportation and organelles clearance of erythroblasts. Clod depletes macrophages in several organs including bone marrow (BM), which destroys murine EBI structure and contributes to significant toxicity due to impaired erythropoiesis.

Our preliminary results indicated the maturation sufficiency of human erythroblasts in HIS mice, as indicated by the hemoglobin switch and potential to differentiate into mature RBCs in vivo. Interestingly. we also observed hybrid EBIs with human erythroblasts and murine macrophage NCG HIS mouse BM, and hypothesized that human EBI reconstitution in HIS mice would improve human erythroblasts development and increase circulating mature hRBCs in vivo. We used a combination of anti-mouse CSF1R and F4/80 antibodies with dexamethasone for repopulating human EBIs in HIS mice (EBI-HIS). Human EBI structure in BM was captured by imaging flow cytometry, which was characterized by human CD14⁺CD163⁺ macrophages surrounded by human CD235a⁺ erythroblasts of varying sizes. Our finding demonstrated that human EBIs reconstitution allowed higher percentages of circulating hRBCs compared to Clod injection, representing up to 14.4% of total RBCs in PB. Notably, hRBCs in PB of EBI-HIS mice closely resemble those in human blood, which show similar hCD71 and hCD235a expression, as well as comparable enucleation rates.

Hereditary MgA is an inherited blood disorder caused by genetic mutations affecting Vitamin B₁₂ and folate absorption. Megaloblasts are prone to destruction in blood vessels, resulting in anemic syndromes of patients. Few mouse models have successfully recapitulated MgA due to its genetic complexity. To determine whether circulating mature hRBCs in EBI-HIS mice would be sufficient to replicate human anemic diseases in vivo, we purified human HSCs from BM samples of hereditary MgA patients and engrafted them into newborn pups. Human erythroblasts in BM showed larger cell size. Abnormal nucleated hRBCs were observed in PB of patients HSCs-engrafted mice but not in normal HSCs-engrafted mice. Tissues analysis of MgA HSC-engrafted mice revealed significant abnormalities, identified by human hemoglobin alpha antibody. Lungs showed increased thrombosis and alveolar hemorrhage, microvascular thromboses and presence of hRBCs in sinusoids was observed in liver samples. Peritubular capillaries and glomeruli engorged were identified in kidneys, vascular occlusion and thrombosis were obvious in spleens of MgA HSCs-engrafted mice but not of normal HSCs-engrafted mice. Thus, EBI-HIS model allows successful establishment of human erythropoiesis and recapitulates pathologic changes of MgA diseases in vivo.

In summary, we herein present our EBI-HIS mice with enhanced human erythropoiesis and circulating fully mature hRBCs. This improved human RBC model recapitulated clinical features of life-threatening erythropoietic disorders like hereditary MgA, and could serve as a valuable preclinical animal model for development and evaluation of therapeutics.

Lodish:Carmine Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Board of Directors; Cerberus: Membership on an entity's Board of Directors or advisory committees, Other: Board of Directors; Paratus Sciences Corporation: Membership on an entity's Board of Directors or advisory committees, Other: Board of Directors; Tevard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Board of Directors. Li:GemPharmatech: Consultancy.