The Assessment of Human Erythroid Output in NOD/SCID Mice Reconstituted with Human Hematopoietic Stem Cells

Abstract

The third-generation NOD/LtSz-scid/IL2Rγnull (NOD/SCID IL2Rγnull) mouse represents a significantly improved xenograft model allowing high levels of human leukocyte engraftment over extended follow up. One remaining limitation of this mouse model, however, is the low level of circulating human erythrocytes. We established a practical ex vivo erythroid culture system of xenograft marrow progenitors to enrich for human erythroid progeny. At various time points after transplant, erythroid cells were easily assayed after 17 days of ex vivo culture of xenograft marrow, with nearly all nucleated cells of human origin and approximately 60% human GPA or CD71 positive. We then transplanted cord blood CD34+ cells marked with a lentiviral vector encoding green fluorescent protein (GFP). Three months later, ex vivo culture of xenograft marrow progenitors showed 41.3% of the cultured erythroid cells were positive for GFP and human CD71, and 56.2% were positive for GFP and human GPA, similar to that of circulating leukocytes at the same time point. Next, G-CSF mobilized peripheral blood CD34+ cells from a sickle cell trait subject were infused in this mouse model to determine if the hemoglobin pattern could be modeled. CD34+ cells from the sickle cell trait subject engrafted equally compared to CD34+ cells from normal subjects, establishing the sickle cell trait phenotype. Lastly, a comparison of adult-derived peripheral blood CD34+ cells and cord blood-derived CD34+ cells xenografted mice was made, and long term follow-up demonstrated a recapitulation of the fetal to adult hemoglobin switch. This approach should prove a useful tool for testing strategies for genetic manipulation of erythroid progeny and the study of hemoglobin switching.

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

“…This suggests that hiPSC-derived erythropoiesis mimics that of human development and recapitulates accordingly the globin gene switch. It also indicates that complete maturation of immature erythroid cells can take place under the influence of an adult hematopoietic microenvironment, as we have already described for cord blood cells, 30 and in accordance with observations of Hayakawa et al 32 that the combination of the murine marrow microenvironment and cytokines is sufficiently potent to mimic the normal post-natal human hemoglobin switching process. We assume that the delay required for the cells to achieve complete hemoglobin switching is linked to their stage of maturation.…”

Macrophages prevent human red blood cell reconstitution in immunodeficient mice

“…24 The failure of human cytokine treatment to improve human RBC reconstitution in humanized mice without macrophage depletion further supports the argument that macrophages pose a critical obstacle to human RBC reconstitution in humanized mice.…”

“…on May 10, 2018. by guest www.bloodjournal.org From This is consistent with a recent study showing that bone marrow recovered from humanized mice could differentiate into human erythroid cells ex vivo. 24 The failure of human cytokine treatment to improve human RBC reconstitution in humanized mice without macrophage depletion further supports the argument that macrophages pose a critical obstacle to human RBC reconstitution in humanized mice.…”

Cord blood endothelial progenitor cells as therapeutic and imaging probes

“…We successfully achieved stable, long-term expression of the transduced GFP gene that was most robust in cells transfected with constructs containing cellular polypeptide chain EF1α and human cytomegalovirus (CMV) promoter [85]. A novel and interesting approach by Hayakawa et al demonstrated the feasibility of long-standing, viral introduced GFP expression in CB CD34 + cells under in vivo condition [86]. Three months after injecting GFP transduced, human CB CD34 + cells into nonobese diabetic/severe combined immunodeficient NOD/SCID IL2R γ null mouse, a significant (33%) percentage of peripheral blood cells were positive for the human CD45 marker, out of which 79.4% were GFP positive.…”

“…Three months after injecting GFP transduced, human CB CD34 + cells into nonobese diabetic/severe combined immunodeficient NOD/SCID IL2R γ null mouse, a significant (33%) percentage of peripheral blood cells were positive for the human CD45 marker, out of which 79.4% were GFP positive. In addition, cells harvested from the bone marrow of mice that received GFP + CB CD34 + cells exhibited GFP expression in ex vivo culture for up to 18 days post-plating [86]. Despite the efforts to optimize and improve techniques based on introducing and stably maintain GFP expression in transduced cells, drawbacks related to GFP detection and imaging still exist.…”