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Identification of phenotypes of human hematopoietic cells that are highly enriched in those with long-term repopulating activity in immunodeficient mice has been essential to elucidating mechanisms that regulate their properties. First trimester human fetal liver (hFL) has long been appreciated as a source of HSCs with remarkable in vivo regenerative capability as compared to their adult or even neonatal counterparts, although conditions to support the in vitro maintenance of this function, particularly of hFL HSCs, have remained elusive. In a first series of experiments, we found that expression of the CD49f integrin on first trimester GPI80+CD90+CD38-CD45RA-CD34+CD45+ hFL cells selectively depleted cells with initial myeloid colony-forming activity in methylcellulose cultures. At the same time, this phenotype became enriched for cells with immediate long-term repopulating capability in both 12-week growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NRG-W41 mice. Next, we compared the functional properties of this enriched HSC subset following their maintenance in vitro for 2 days under different culture conditions. The results demonstrated that FLT3-L, as a single GF in serum-free medium best maintained their ability to regenerate serially transplantable HSCs. In contrast, the same 2-day pre-transplant culture condition resulted in the poorest survival and division of the input cells. Subsequent cell cycle analysis confirmed the existence of a G0 population within the HSC-enriched subset of hFL cells that best maintains their growth potential in FLT3-L alone. These results demonstrate CD49f expression to be a pervasive marker of human HSCs in vivo and reinforce the concept that the maintenance of HSC properties may be adversely affected by factors that strongly promote survival and rapid cell cycle entry. [ABSTRACT FROM AUTHOR]

Differences between mice and humans, such as variations in the major histocompatibility complex (MHC) and variances in immune system function, pose challenges to accurately model human biology in mice. Therefore, improving humanized mouse models, particularly models of human hematopoietic lineages (such as functional lymphocytes), is essential. This optimization is especially relevant to the study of the immune system for advancing treatments like CAR-T therapy and immune checkpoint inhibitors in cancer treatment. A novel mouse model with the essential immune components needed to generate mature B and T cells has been developed. The DRAGA mouse model has an NRG background and carries human HLA-A2 and HLA-DR4 transgenes. These transgenes have been shown to enhance hematopoietic engraftment and promote the development of fully functional T cells and antibody-secreting B cells (Casares, 2016). To test the ability of the DRAGA mouse strain to support the late-stage differentiation of human B- and T-cells from HLA-matched cord blood (CB) progenitors, we transplanted 10,000 matched or unmatched CD34+ CB cells into DRAGA mice. We then tracked the output of human hematopoietic cells for 20 weeks. Analysis showed higher T- and B-cell progenitors in the bone marrow of mice engrafted with matched cord blood progenitors compared to unmatched ones, indicating that HLA compatibility may enhance mature T-cell selection and early lymphopoiesis. In addition, comparing matched cord blood progeny with infant human thymus tissue reveals similar T-lineage phenotypes, suggesting this model replicates this aspect of human T cell development and maturation in mice. Our findings suggest that the DRAGA mouse holds promise as a model for creating a humanized immune system. This model may be valuable for studying immunotherapies and exploring immune cell functions across different cancer types. [ABSTRACT FROM AUTHOR]

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice. [ABSTRACT FROM AUTHOR]

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34 + CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells. [ABSTRACT FROM AUTHOR]